The practical utilization of regenerative medicine, which helps the regeneration of living tissues/organs that have fallen into functional disorder or functional incompetence, is currently proceeding. The regenerative medicine is novel medical technology of re-creating the same or similar forms or functions as in original tissues using 3 factors, i.e., cells, scaffolds, and growth factors, for living tissues that no longer recover by only natural healing ability intrinsically possessed by organisms. In recent years, treatments using cells have been being gradually realized. Examples thereof include cultured epidermis using autologous cells, cartilage treatment using autologous cartilage cells, bone regeneration treatment using mesenchymal stem cells, cardiac muscle cell sheet treatment using myoblasts, corneal regeneration treatment using corneal epithelial sheets, and nerve regeneration treatment. These novel treatments, unlike conventional alternative medicine based on artificial materials (bone prosthetic materials or hyaluronic acid injection), help the repair or regeneration of living tissues and therefore produce high therapeutic effects. In fact, some products such as cultured epidermis or cultured cartilage using autologous cells have been launched.
However, current techniques cannot provide tissues having a sufficient thickness, because cells to be transplanted are mainly transplanted in a thin sheet form or transplanted in the state of a suspension. Living tissues are originally thick and enable muscle force to allow the heart to beat or permit smooth movement at articular cartilage because of being thick. For general tissue regeneration using cells, the inability to provide thick tissues is considered as a major problem.
For example, the regeneration of cardiac muscle using cell sheets is considered to require a multilayer construct of cell sheets for regenerating thick tissues. Okano et al. have recently developed cell sheets using a temperature-responsive culture dish. The cell sheets do not require treatment with an enzyme such as trypsin and thus retain cell-to-cell binding and adhesion proteins (Non Patent Documents 1 to 6). Such a cell sheet production technique is expected to be useful in the regeneration of cardiac muscle tissues. Since previous cell sheets cannot form vascular network, sufficiently thick tissues have been difficult to regenerate (Non Patent Documents 5 and 7). This is because nutrition supply to cells in the central portion is lost in a cell sheet which was allowed to be thick, whereby the cells are killed. Okano et al. have also thought that a thickness of 200 μm or larger is impossible to achieve, and are developing cell sheets also containing vascular endothelial cells introduced therein in order to form vascular network in the cell sheets (Non Patent Document 8). However, this cannot serve as a realistic solution due to many problems: in addition to the cells of interest, another cell source, i.e., vascular endothelial cells, must be prepared; it is difficult to uniformly induce blood vessels in the cell sheet; and even if the delivery pathway of nutrients can be provided by this means, the prepared nutrition delivery pathway must be precisely connected to an external nutrition delivery pathway in this approach.
Also for bone regeneration, bone regeneration sheets comprising cultured cells added to matrices have been developed.
A bone regeneration sheet prepared by layering a cultured cell sheet comprising mesenchymal stem cells cultured into a sheet-like shape and a biodegradable sheet comprising biodegradable substances formed into a sheet-like shape (Patent Document 1) has been proposed. Moreover, there is a sheet for induction of mesenchymal tissue regeneration in which mesenchymal tissue precursor cells differentiated from mesenchymal cells and extracellular matrices are attached onto a porous sheet (Patent Document 2). These inventions are methods involving placing a cultured osteoblast-attached sheet into the body and forming cortical bone from the osteoblast through membranous ossification in vivo. However, osteoblast-like cells cannot be cultured in a layered state, and due to this problem, sheets having an osteoblast layer have failed to provide regeneration sheets in which the thickness of a cell layer exceeds 100 μm. Then, Patent Document 3 has reported that a sheet of 200 μm or larger in thickness can be formed by the development/optimization of a culture approach, but according to this report, only the formation of approximately 210 μm cortical bone tissue layer was achieved.
As described above, it was a difficult challenge to provide cells as a thick composition for many tissue repairs. The leading cause thereof is the insufficient penetration of nutrients by only diffusion into a three-dimensional construct composed of cells. Gel-embedding culture using collagen has been devised as one means of solving this (Non Patent Document 9). However, cells embedded in a gel cannot solve this problem at its source, because the cells are moved from the central portion of the gel toward the outer region and thus, are not uniformly present in the gel so that the cell density of the central portion is reduced. Moreover, the three-dimensional cell construct prepared by gel embedding cannot be bound/fused to another three-dimensional construct and thus, cannot form a three-dimensional construct above the size prepared at the time of cell inoculation. Thus, the means of preparing small gels and then fusing the gels to each other to prepare a construct in which cells are uniformly distributed, cannot be adopted.
Moreover, Patent Document 4 states that three-dimensional culture is achieved by linking cells using inorganic ceramic beads. However, inorganic ceramics are inferior in water retention, solution exchange, diffusion of nutrition, and buffer capacity and cannot actually provide a thick cell composition. In fact, in Examples of Patent Document 4, cells were bonded to 150 to 460 μm particles, over which a thick PLLA nonwoven fabric (1 cm) was layered to merely increase an apparent thickness. The actual cell-containing layer was merely a layer of tens of μm at the thickest on the surface of the inorganic ceramic beads. Even if the 1 cm PLLA nonwoven fabric having no cell is regarded as a construct, it is merely a construct having significantly nonuniform cell distribution. Thus, only the three-dimensional cell construct having nonuniform cell distribution in the construct or the construct having a substantially thin cell layer has been provided so far.